1. Technical Field
The present disclosure is directed to advantageous apparatus, systems and methods for obtaining desired measurements. More particularly, the present disclosure is directed to apparatus, systems and methods for measuring properties of biomolecules, e.g., proteins. The disclosed apparatus, systems and methods permit simultaneous measurement of scattered light intensities and concentration, e.g., using a single flow cell. Moreover, the disclosed apparatus, systems and methods permit accurate measurements to be made that support and/or facilitate calculation of the thermodynamic second viral coefficient of molecules, while simultaneously addressing issues associated with interdetector delay volume (IDV) and/or band-broadening. Exemplary embodiments of the present disclosure facilitate protein-related measurements using an advantageous dual-detector cell, and the dual-detector cell may be employed in the determination of the second viral coefficient of proteins in aqueous solutions.
2. Background Art
Protein-protein interactions play an important role in several phenomena of interest, including protein crystallization (George et al., 1997; George and Wilson, 1994), which relates to protein solubility (Guo et al., 1999; Rosenbaum and Zukoski, 1996), amorphous precipitation (Curtis et al., 2002; Piazza, 1999; Poon, 1997), formation of reversible protein aggregates in supersaturated solutions (Knezic et al., 2004), and irreversible aggregation (Chi et al., 2003a; Ho et al., 2003; Petsev et al., 2000; Zhang and Liu, 2003). These in turn have implications in the pathology of diseases, such as Alzheimer's (Fabian et al., 1993) and in the stability of protein pharmaceuticals (Chi et al., 2003b).
The second viral coefficient, B22, which is a result of protein-protein interactions, represents non-ideality in dilute protein solutions (Tanford, 1961), and has been widely used as a parameter to study weak protein-protein interactions in aqueous solutions, e.g., to indicate whether the protein molecules (macromolecules, biomolecules) are experiencing a net attractive force or a net repulsive force. For example, correlation has been shown among B22 values, solubility of proteins, and solution conditions under which protein crystals can be obtained (Guo et al., 1999).
Widespread application of B22 values for investigating protein-protein interactions is lacking, presumably due to the limitations of the commonly employed techniques of batch-mode static light scattering, membrane osmometry, and sedimentation equilibrium. In addition to the long time durations necessary to complete these experiments (˜1-2 days), these techniques require large amounts of protein (˜25-100 mg) in order to obtain reliable estimates for B22 values. Furthermore, errors can be introduced from impurities in the sample, such as dust particles or protein aggregates.
Recently, reports have emerged on rapid and improved methods to estimate protein-protein interactions in aqueous solutions, i.e., methods based either on self-interaction chromatography (Tessier et al., 2002) or size-exclusion chromatography (Bloustine et al., 2003). Although promising, these techniques require additional steps for determination of the B22 values. The technique of self-interaction chromatography, for example, requires prior immobilization (Tessier et al., 2002) of the same protein; unfortunately, immobilization itself can affect protein structure and, hence, protein-protein interactions. Attempts to utilize size-exclusion chromatography (SEC) (Bloustine et al., 2003), which is routinely used in protein molecular weight characterization, have also been described for the measurement of protein-protein interactions. Bloustine et al. (2003) utilized the solute distribution coefficient as determined from the retention times in SEC to obtain the B22 values of proteins in aqueous solutions, and Wyatt (2002) recently disclosed the use of SEC utilizing a light scattering detector and a concentration detector connected in series to obtain the B22 values of proteins. Although this technique minimizes contributions from dust and aggregate impurities, it is still prone to errors arising from interdetector delay volume (IDV) and interdetector band broadening (Netopilik, 1997, 2003; Shortt, 1994; Wyatt, 1993b; Wyatt and Papazian, 1993; Zammit et al., 1998) within the two detectors, and hence requires mathematical correction factors to obtain the B22 values.
The IDV and band broadening issues in SEC utilizing two detectors (i.e., a light scattering detector and a concentration detector, such as an ultraviolet detector) connected in series are significant, especially when discrete data points on the chromatogram, rather than the whole chromatogram, are used for analysis. When the protein sample, after separation in the SEC column, passes through the two detectors in series, a lag time occurs in the chromatogram due to physical separation of the detectors that relates to IDV. For proper analysis, the chromatograms from the two detectors must be overlaid precisely after correcting for this IDV. This is commonly attempted by measuring the peak-to-peak time difference between the two chromatograms, using a known standard and converting this time difference to the IDV from flow rate information. Once known, this IDV is then used for all samples. The IDV phenomenon is schematically represented in FIG. 1B, which is described in greater detail hereinafter.
U.S. Pat. No. 5,305,071 to Wyatt discloses a refractometer structure. The disclosed refractometer includes a capillary, is surrounded by detectors coplanar with the capillary, and is illuminated through the capillary by a light source, such that the angular variation of light scattered by particles flowing through the capillary is measured. A second light source and a displacement detector are incorporated into the apparatus such that the refractometer also functions as a concentration sensitive detector. According to the Wyatt '071 patent, the disclosed refractometer, when combined with the technique of size exclusion chromatography, will permit measurements of molecular size and weight of each separated fraction irrespective of the constancy of flow rate, since both light scattering and concentration measurement may be performed on the same flowing volume element of effluent. The Wyatt '071 patent further states that the disclosed refractometer, when used with another form of concentration detector, such as ultraviolet absorption or evaporative mass detection, will permit deduction of the differential refractive index increment dn/dc with concentration and that, in this manner, the physical parameters of co-polymers may be derived by combining the measurements of the differential refractometer, light scattering array, and concentration sensitive detector.
U.S. Pat. No. 6,404,493 to Altendorf discloses a dual large angle light scattering detection device/system with a configuration that is particularly suitable for use with planar liquid sample flow cells. The analyzer includes a polarized light source and at least two large angle scattered light photodetectors positioned at acute and right (or oblique) angles to the incident light beams, respectively. Differences in intensities of light measured at the two photodetectors are used to quantify components of the sample
U.S. Pat. No. 4,693,602 to Wyatt et al. provides a system for measurement of the scattering properties of very small particles by electro-optical means. The Wyatt '602 system generally requires the use of an intense, though highly spatially inhomogeneous, light source such as a laser. The absolute intensity of the light incident on the particle need not be known. A special structure and measurement process are described by which small particles are differentiated from larger particles grazing the illumination beam.
U.S. Pat. No. 5,530,540 to Wyatt et al. provides a modified light scattering cell, and associated method, whereby an eluant of very small dimension transverse to its direction of flow is entrained successively by two sheath flows and presented to a fine light beam that illuminates the entrained eluant as it flows through the light beam. The light scattered by the entrained eluant is collected by detectors outside of a transparent flow cell enveloping the sheath flow entrained eluant. The windows of the transparent flow cell through which the light beam enters and leaves are far removed from the scattering eluant and kept clear of eluant-contained particles by means of flow components that will form subsequently one of the eluant sheath flows employed. The eluant source is typically from a fine capillary such as found in capillary electrophoresis, capillary hydrodynamic fractionation, and flow cytometry applications.
U.S. Pat. No. 6,651,009 to Trainoff et al. provides a method for measuring the molecular properties of an unfractionated solution of macromolecules. Sharing some similarities with the standard Zimm plot technique, the method begins with the preparation of several sample aliquots spanning a range of concentrations. The aliquots are then injected sequentially into a stream such as provided by a liquid chromatograph. Each aliquot produces an effective “peak” whose elements correspond to different concentrations of the diluted aliquot. By analyzing the angular and concentration dependence of the scattering signals throughout the corresponding peaks, the weight averaged molar mass, the mean square radius, and the second viral coefficient may be derived.
U.S. Pat. No. 6,411,383 to Wyatt provides a method for determining the 2nd viral coefficient of an ensemble of molecules dissolved in a selected solvent. Two distinct classes are described: monodisperse and polydisperse molecules. If the molecules are monodisperse, the Wyatt '383 patent teaches that they must be prepared for a chromatographic separation and suitable columns selected. Following standard chromatographic separation procedures, such as exemplified by the method of size exclusion chromatography, the sample passes through the separation columns, a multi-angle light scattering detector, and a concentration detector. The effect of the columns is to produce a concentration profile of the sample that appears as a peak as it passes through the light scattering and concentration detectors. For each elution interval, vi, a corresponding concentration value ci and set of excess Rayleigh ratios Ri (Θj) is measured for each scattering angle Θj. The excess Rayleigh ratios are extrapolated to Θ=0° resulting in the calculation of a single extrapolated value for each elution slice, viz., Ri (0°). Three sums are calculated from the data collected: 1) the sum of all ci values over the measured concentration peak; 2) the sum of all (ci)2 values over the same concentration peak; and 3) the sum of all the extrapolated Rayleigh ratios over the measured light scattering peak. According to the Wyatt '383 patent, the 2nd viral coefficient is calculated directly from these three quantities once the molecule's molar mass is known. The same procedure is followed for polydisperse samples; however, the column set is replaced by a dilution means that does not fractionate the sample.
U.S. Pat. No. 5,676,830 to Janik et al. discloses a modified capillary tube used to transfer a liquid sample into a detection cell following separation by a chromatographic system. The capillary tube is modified by plugging or otherwise severely restricting its flow. Near its plugged end, the tube is drilled to provide a plurality of holes or ports perpendicular thereto and penetrating into the central flowing core of the tube so as to direct outflow from the tube perpendicularly therefrom. The outer diameter of this modified capillary tube is selected to be of a size comparable to, though smaller than, the detection cell diameter into which it transfers the flowing sample. In this manner, fluid transferred into a detection cell by the modified capillary tube will be split into a plurality of smaller streams flowing outwardly therefrom and striking the adjacent detector cell walls almost immediately. Because of the close proximity of the emerging split streams to the walls of the detection cell, the eddies produced thereby will be very small and the contents of the detection cell will be homogenized rapidly.
U.S. Pat. No. 4,616,927 to Phillips et al. provides a sample cell that permits measurement of the light scattering properties of very small liquid-borne samples with negligible background interference from the illumination source. The cell construction permits the measurement of illumination intensity at the scattering sample itself, thereby permitting normalization of each detected scattered signal. The cell structure and detection method incorporated therein also permit measurement of extremely small angle-scattered intensities without interference of the incident light beam itself.
U.S. Pat. Nos. 5,250,186 and 5,269,937 to Dollinger et al. describe a high angle light scattering detector using classical Rayleigh scattering. A high intensity arc light source, filtered to leave only one wavelength, illuminates a flow cell. Through the flow cell, very small particles (such as biological proteins) flow in solution after separation by HPLC or some other means. A UV detector generates data regarding the weight concentration of the eluting particles and a scattered light detector collecting scattered light at angles of approximately 90° generates a scattered light signal. The incident light intensity is also measured. The average molecular weight is then computed using the scattered and incident light data, the weight concentration data and a simplified mathematical relationship from which the size factor P and the viral coefficients have been eliminated.
Despite efforts to date, a need remains for advantageous systems and methods for measuring properties of biomolecules, e.g., proteins. In addition, a need remains for systems and methods that permit simultaneous measurement of light scattered intensities and concentration, e.g., using a single flow cell. Moreover, there is a continuing need for systems and methods that permit accurate measurements to be made that support and/or facilitate calculation of the thermodynamic second viral coefficient of molecules, while simultaneously addressing issues associated with IDV and/or band-broadening. These and other needs are satisfied by the apparatus, systems and methods disclosed herein.